Ghaffari lab studies mechanisms that sustain blood forming stem and progenitor cell production throughout life and that are perturbed in disease. We are particularly interested in programs that maintain stem cell quiescence, a property that determines the potency and overall regenerative capacity of adult stem cells and that is lost with age. Quiescence is also a mechanism by which malignant stem cells resist therapy. To attain this goal, we have been investigating mitochondrial-regulated programs in hematopoietic stem and progenitor cells. We are using a variety of approaches including various omics, imaging and more recently gene editing technology combined with genetically modified mouse models and human cells to address these questions. Our studies are conducted in the physiological context of primary cells in vivo and in vitro under homeostasis and in disease.

Old blood in a young body

Hematopoiesis, Hematopoietic Stem Cells and Erythropoiesis

Blood-forming stem cells have unique properties that enable them to live a long life and regenerate blood constantly. The well-being of blood-forming stem cells throughout life is essential for maintaining a healthy life by producing billions of red and white blood cells every day in humans. With aging this regenerative capacity declines which compromises optimum blood production and might also lead to blood malignancies whose incidence increases with age. The primary goal of our laboratory is to dissect the regulatory pathways that maintain the health of blood-forming stem and progenitor cells throughout life. We hope this knowledge will eventually lead to the identification of specific targets and/or development of novel tools that could be used in the clinic to improve therapy and/or to produce healthy blood-forming stem cells in a dish for bone marrow transplantation. In these efforts, we have identified the longevity transcription factor FOXO3 and its regulatory network as critical protector of blood-forming stem cells health and driver of stem cell production of red blood cells. We are currently investigating FOXO3-mediated and independent mitochondrial-regulated programs in stem cells and in erythroid cells.

Red Blood Cells and Erythropoiesis

Erythropoiesis is the process of making red blood cells (RBCs) from hematopoietic stem and progenitor cells. RBCs carry oxygen and travel throughout the body to oxygenate all tissues. Anemia or reduced capacity of RBCs to carry oxygen constitutes a major health problem associated with many disorders. In response to loss or reduced production of RBCs, blood-forming stem and progenitor cells are activated to produce RBCs. RBC production is dependent mainly on erythropoietin, a hormone that is produced when oxygen levels are low. We found FOXO3, a protein that regulates a response to oxygen levels and whose function is partly controlled by erythropoietin, as key to

Figure 1

physiological regulation of red blood cell maturation and production and the regulation of harmful consequences of oxygen metabolism (oxidative stress) during this process (Marinkovic et al., JCI, 2007). We recently discovered that a fascinating feature of FOXO3 regulation of RBC production is its control of the essential enucleation (removal of the nucleus) process in coordination with mitochondrial clearance through autophagy (mitophagy) (Liang et al., PLoS Genetics, 2015). We are currently investigating how mitochondria and nucleus communicate in erythroid cells and the contribution of FOXO3 to this process.

Hematopoietic Stem Cells and Mitochondria

Quiescence is a fundamental property of most adult stem cells. The regeneration capacity of adult blood-forming stem cells is tightly linked to their quiescence, a property that requires the transcription factor FOXO3. Mitochondria are the center of energy production and critical for blood-forming stem cell activation. One of the byproduct of mitochondrial activation is the generation of reactive oxygen species (ROS) that depending on the levels and/or context might be deleterious (oxidative stress). Cumulative findings raise the possibility that mitochondria are critical for blood-forming stem cell activity although very little is known about mitochondria in adult blood-forming stem cells. Our work indicates that FOXO3 is required for regulating both ROS levels and mitochondrial metabolism in blood-forming stem cells possibly through independent mechanisms (Rimmelé et al., EMBO Reports, 2015). We are delving deep into mitochondrial-regulated programs in blood-forming stem cells. We are aiming to identify molecules that abrogate mitochondrial remodeling that occurs with stem cell alteration to be used therapeutically.

Figure 2

Hematopoietic Stem Cells Aging, and SIRT1 regulation of FOXO3

Aging of long lived blood forming stem cells leads to the accumulation of damaged DNA and proteins overtime which reduces their regenerative capacity and promotes their potential to develop malignancy. Specifically, aging of blood-forming stem cells is thought to be at the origin of myeloid leukemias and myeloproliferative neoplasms (MPNs) whose incidence increase with age. We recently discovered that the loss of the NAD dependent deacetylase Sirtuin (SIRT1) in hematopoietic stem cells mimics closely the aging phenotype of blood forming stem cells (Rimmelé et al., Stem Cell Reports, 2014). We further found that SIRT1 effects in hematopoietic stem cells are partially mediated by FOXO3. Our findings raise the possibility that SIRT1 regulation of FOXO3 may constitute a program that protects hematopoietic stem cells from age-associated damage. We are currently investigating this hypothesis.

Figure 3

Leukemic Stem Cells Reprogramming

Leukemic stem cells are thought to be rare cells that have stem cell properties, are quiescent and resist therapy, and reestablish the disease sometimes years after remission. Understanding the biology of leukemic stem cells may provide unique therapeutic opportunities. Although FOXO3 is clearly implicated in myeloproliferative neoplasms (MPNs) and acute myeloid leukemia (AML) whether FOXO3 protects or enhance leukemogenesis remains unclear. In collaboration with other laboratories at ISSMS, United States and abroad we are addressing FOXO3 regulation and function in the context of myeloid malignancies.